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Characterization of Eutypa Lataand Cytospora Pistaciaecausing

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Characterization of Eutypa Lataand Cytospora Pistaciaecausing Phytopathologia Mediterranea Firenze University Press The international journal of the www.fupress.com/pm Mediterranean Phytopathological Union New or Unusual Disease Reports Characterization of Eutypa lata and Cytospora pistaciae causing dieback and canker of Citation: D. Aiello, G. Polizzi, G. Gusella, A. Fiorenza, V. Guarnaccia pistachio in Italy (2019) Characterization of Eutypa lata and Cytospora pistaciae causing die- back and canker of pistachio in Italy. Phytopathologia Mediterranea 58(3): Dalia AIELLO1,*, Giancarlo POLIZZI1, Giorgio GUSELLA1, Alberto 699-706. doi: 10.14601/Phyto-10880 FIORENZA1, Vladimiro GUARNACCIA2,3 Accepted: September 26, 2019 1 Dipartimento di Agricoltura, Alimentazione e Ambiente, sezione Patologia Vegetale, University of Catania, Via S. Sofia 100, 95123 Catania, Italy Published: December 30, 2019 2 Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Nether- Copyright: © 2019 D. Aiello, G. Poliz- lands 3 zi, G. Gusella, A. Fiorenza, V. Guar- DiSAFA, University of Torino, Largo Paolo Braccini, 2, 10095 Grugliasco, TO, Italy naccia. This is an open access, peer- *Corresponding author: [email protected] reviewed article published by Firenze University Press (http://www.fupress. com/pm) and distributed under the Summary. During the winter of 2017, dieback and canker symptoms were observed terms of the Creative Commons Attri- on pistachio (Pistacia vera) in two orchards in the Bronte area, Catania Province, Sic- bution License, which permits unre- ily, Southern Italy. Two different fungi were consistently isolated from infected tissues. stricted use, distribution, and reproduc- Morphological observations and multi-locus phylogenies using five genomic loci (ITS, tion in any medium, provided the origi- act, rpb2, tef1 and tub2) identified these fungi as Cytospora pistaciae and Eutypa lata. nal author and source are credited. Pathogenicity tests on 5-y-old potted plants of P. vera grafted on terebinth P.( terebin- Data Availability Statement: All rel- thus) reproduced similar symptoms as those observed in nature, and Koch’s postulates evant data are within the paper and its were fulfilled for these two pathogens. This study is the first to report dieback and can- Supporting Information files. ker diseases of pistachio caused by C. pistaciae and E. lata in Italy. Competing Interests: The Author(s) Keywords. Pathogenicity, molecular analysis, disease symptoms, Pistacia vera. declare(s) no conflict of interest. Editor: Lizel Mostert, Faculty of AgriSciences, Stellenbosch, South Africa. INTRODUCTION Pistachio is cultivated in the southern regions of Italy, of which Sicily is the main production area. The province of Catania (with 430 ha of pista- chio), followed by the provinces of Caltanissetta (with 220 ha) and Agrigento (with 145 ha) are the largest pistachio-producing areas, with a total produc- tion of 3,878 tons (AGRISTAT, 2017). Currently, the commune of Bronte in Catania Province represents the most important area of Sicily for pistachio production, and pistachio is an important economic resource for this terri- tory (Barone and Marra, 2004). In this area, different pistachio cultivars are grafted on terebinth plants which are grown on volcanic soils (Barone et al., 1985). Few studies have been conducted to investigate pistachio diseases occurring in Italy, and only a few diseases have been reported to date. These include branch dieback (caused by Botryodiplodia sp.), leaf spot (Alternaria alternata), anthracnose, branch and twig cankers (Botryosphaeria dothidea) Phytopathologia Mediterranea 58(3): 699-706, 2019 ISSN 0031-9465 (print) | ISSN 1593-2095 (online) | DOI: 10.14601/Phyto-10880 700 Dalia Aiello et alii and phylloptosis and leaf spots (mainly caused by Septo- DNA extraction, PCR amplification and sequencing ria pistaciae) (Casalicchio, 1963; Schilirò and Privitera, 1988; Frisullo et al., 1996; Vitale et al., 2007). In east- Extractions of genomic DNA were performed from ern Sicily, cankers and decline caused by Liberomyces pure cultures, as reported elsewhere (Guarnaccia and pistaciae Voglmayr, Vitale, Aiello, Guarnaccia, Luongo Crous, 2017), using the Wizard Genomic DNA Purifica- & Belisario are the most important pistachio diseases tion Kit (Promega Corporation). Partial regions of five (Vitale et al., 2018). Blight caused by Arthrinium xeno- loci were amplified. The primers ITS5 and ITS4 (White cordella Crous was also recently reported on pistachio et al., 1990) were used to amplify the internal tran- fruit in the Agrigento Province (Aiello et al., 2018). scribed spacer region (ITS) of the nuclear ribosomal During the winter of 2017, pistachio trees with die- RNA operon, including the 3’ end of the 18S rRNA, the back, canker and gummosis symptoms were observed first internal transcribed spacer region, the 5.8S rRNA in the area of Bronte. Following culturing from necrot- gene; the second internal transcribed spacer region and ic tissues, two fungal species were consistently isolated. the 5’ end of the 28S rRNA gene. The primers ACT- Cankers from one orchard generated colonies of Cyto- 512F and ACT-783R (Carbone and Kohn, 1999) were spora while cankers from a second orchard generated used to amplify part of the actin gene (act). The partial Eutypa colonies. beta-tubulin (tub2) gene was amplified with primers Bt- The aim of the present study was to investigate the 2a and Bt-2b (Glass and Donaldson, 1995). The prim- etiology of pistachio canker diseases, which could repre- ers EF1-728F and EF1-986R (Carbone and Kohn, 1999) sent new threats for the pistachio production of Sicily. were used to amplify part of the translation elongation factor 1-α gene (tef1). The primers 5f2/7cr were used to amplify part of rpb2 (O’Donnell et al., 2010). The regions MATERIALS AND METHODS ITS, act, tef1 and rpb2 were amplified for the species of Cytospora using the PCR programmes adopted by Law- Isolation and morphology of fungi rence et al. (2018) and Jami et al. (2018). The regions ITS and tub2 were amplified for the species of Eutypa follow- Surveys were conducted in ten pistachio orchards ing the PCR programmes used by Moyo et al. (2018a). with histories of branch canker and dieback in east- The PCR products were sequenced in both directions ern Sicily (Catania Province). Approximately 20 using the BigDye® Terminator v. 3.1 Cycle Sequencing symptomatic pistachio branches with canker were col- Kit (Applied Biosystems Life Technologies), after which lected from each orchard for analyses. Sub-cortical amplicons were purified through Sephadex G-50 Fine and wood fragments (about 5 × 5 mm) were cut from columns (GE Healthcare) in MultiScreen HV plates the margins between affected and healthy branch tis- (Millipore). Purified sequence reactions were analyzed sues. Tissue pieces were disinfected in 1.2% sodium on an Applied Biosystems 3730xl DNA Analyzer (Life hypochlorite for 60 s, rinsed in sterile water and Technologies). The DNA sequences generated were ana- dried on sterile filter paper. The fragments were then lyzed and consensus sequences were computed using the placed into Petri plates containing potato dextrose program SeqMan Pro (DNASTAR). agar (PDA, Oxoid) amended with 100 mg L-1 of strep- tomycin sulfate (Sigma-Aldrich), and incubated at Phylogenetic analyses room temperature (25 ± 5°C). Fungal colonies consist- ently growing from symptomatic tissues were cultured Novel sequences generated in this study were blast- into new PDA plates. To obtain pure cultures, single- ed against the NCBIs GenBank nucleotide database, to conidium or hyphal-tip isolations were performed determine the closest relatives to be included in the phy- after 1 month incubation at room temperature under logenetic analyses. Blast analyses indicated that three natural light conditions. Isolates for each putative isolates belonged to Cytospora and the remaining four fungal pathogen (four isolates of Eutypa and three to Eutypa. Sequence alignments of the different gene of Cytospora) were characterized by morphological, regions, including sequences obtained from this study molecular and phylogenetic analyses (Table 1). These and sequences from GenBank, were initially performed cultures were deposited in the working collection of using the MAFFT v. 7 online server (http://mafft.cbrc. Dr Pedro Crous (CPC), at the Westerdijk Fungal Bio- jp/alignment/server/index. html) (Katoh and Stand- diversity Institute, Utrecht, The Netherlands (Table 1). ley, 2013), and then manually adjusted in MEGA v. 7 Size and shape of conidia were recorded for each fun- (Kumar et al., 2016). To establish the identity of the fun- gal isolate grown on PDA for 2 weeks at 25 ± 1°C. gal isolates, phylogenetic analyses were conducted using Diseases of pistachio in Italy 701 one locus (data not shown) as well as concatenated anal- showed dieback. Under the bark of affected branch- yses of four loci (ITS, act, tef1 and rpb2) for Cytospora es, cankers were characterized by discolouration and spp. and two loci (ITS and tub2) for Eutypa spp., as indi- necrosis, and in some cases discolouration extended to cated by blast analysis. Additional reference sequences the vascular tissue (xylem) and pith. Two different fun- were selected based on recent studies on Cytospora and gal colony types were consistently obtained from isola- Eutypa species (Lawrence et al., 2018, Moyo et al., 2018a, tions from symptomatic tissues (Figure 1) taken from b). Phylogenetic analyses were based on Maximum the two orchards. Cankers from one orchard generated
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